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Cellular/MolecularCa2ϩ-Dependent Facilitation of Ca v1.3Ca2ϩChannels by Densin and Ca2ϩ/Calmodulin-Dependent Protein Kinase II Meagan A.Jenkins,1*Carl J.Christel,2*Yuxia Jiao,4Sunday Abiria,4Kristin Y.Kim,2Yuriy achev,3Gerald J.Obermair,5Roger J.Colbran,4and Amy Lee21Department of Pharmacology,Emory University,Atlanta,Georgia30322,Departments of2Molecular Physiology and Biophysics and3Pharmacology, University of Iowa,Iowa City,Iowa52242,4Department of Molecular Physiology and Biophysics,Vanderbilt University,Nashville,Tennessee37232,and5Department of Physiology and Medical Physics,Innsbruck Medical University,A-6020Innsbruck,AustriaCa v1(L-type)channels and calmodulin-dependent protein kinase II(CaMKII)are key regulators of Ca2ϩsignaling in neurons.CaMKII directly potentiates the activity of Ca v1.2and Ca v1.3channels,but the underlying molecular mechanisms are incompletely understood. Here,we report that the CaMKII-associated protein densin is required for Ca2ϩ-dependent facilitation of Ca v1.3channels.While neither CaMKII nor densin independently affects Ca v1.3properties in transfected HEK293T cells,the two together augment Ca v1.3Ca2ϩcurrents during repetitive,but not sustained,depolarizing stimuli.Facilitation requires Ca2ϩ,CaMKII activation,and its association with densin, as well as densin binding to the Ca v1.3␣1subunit C-terminal domain.Ca v1.3channels and densin are targeted to dendritic spines in neurons and form a complex with CaMKII in the brain.Our results demonstrate a novel mechanism for Ca2ϩ-dependent facilitation that may intensify postsynaptic Ca2ϩsignals during high-frequency stimulation.IntroductionCaMKII is a serine-threonine protein kinase that is activated by postsynaptic elevations in Ca2ϩand plays a central role in synap-tic plasticity(Hudmon and Schulman,2002;Lisman et al.,2002; Colbran and Brown,2004;Griffith,2004).Ca v1channels mediate L-type Ca2ϩcurrents that can regulate CaMKII activation in dendritic spines(Lee et al.,2009),propagation in dendrites(Rose et al.,2009),and coupling to gene transcription(Wheeler et al., 2008)and synaptic plasticity(Yasuda et al.,2003;Lee et al.,2009). Functional interactions of Ca v1and CaMKII may involve tether-ing of CaMKII to the Ca v1channel complex.CaMKII binds to and phosphorylates the main Ca v1.2␣1(Hudmon et al.,2005) and auxiliary␤subunits(Grueter et al.,2006,2008).These inter-actions augment cardiac Ca v1.2currents in a feedback process known as Ca2ϩ-dependent facilitation(CDF)(Dzhura et al., 2000;Wu et al.,2001;Hudmon et al.,2005;Grueter et al.,2006). CaMKII also causes voltage-dependent facilitation(VDF)of Ca v1.2currents in response to strong depolarizations(Lee et al., 2006).Due to their distinct biophysical properties(Koschak et al., 2001;Scholze et al.,2001;Xu and Lipscombe,2001;Helton et al., 2005),Ca v1.3and Ca v1.2may play different roles in neurons (Calin-Jageman and Lee,2008).Ca v1.3channels regulate spon-taneous firing of substantia nigra dopaminergic neurons(Chan et al.,2007;Puopolo et al.,2007),upstate potentials in striatal medium spiny neurons(Olson et al.,2005),and processes under-lying fear conditioning(McKinney and Murphy,2006)and depression-like behavior(Sinnegger-Brauns et al.,2004).There-fore,Ca v1.3modulation by CaMKII and other factors may have evolved to meet the unique signaling demands of this channel in neurons.Although insulin-like growth factor1can potentiate Ca v1.3currents by a mechanism that requires Ca2ϩrelease from intracellular stores and CaMKII activity(Gao et al.,2006b),the role of CaMKII in direct feedback regulation of Ca v1.3channels is unknown.Here,we describe an unexpected mechanism for Ca v1.3 modulation by CaMKII involving the PDZ[postsynaptic den-sity-95(PSD-95)/Discs large/zona occludens-1(ZO-1)]domain-containing protein densin.Densin is a member of the leucine-rich repeat and PDZ-domain-containing proteins(Bilder et al.,2000) and is localized in the postsynaptic density(Apperson et al.,1996) and binds to and is phosphorylated by CaMKII(Strack et al., 2000;Walikonis et al.,2001).Densin may scaffold other postsyn-aptic proteins,including Shank(Quitsch et al.,2005),␦-catenin (Izawa et al.,2002),and MAGUIN(Ohtakara et al.,2002),and promotes branching of dendrites in cultured neurons(Quitsch et al.,2005).We showed previously that the related protein erbin enhances voltage-dependent facilitation of Ca v1.3(Calin-Jageman et al.,2007).Like erbin,densin binds to the C terminus (CT)of the Ca v1.3␣1subunit but alone does not affect Ca v1.3 properties.When coexpressed with CaMKII,densin facilitates Ca v1.3Ca2ϩcurrents during high-frequency stimulation.Densin and Ca v1.3are targeted to dendritic spines and together associate with CaMKII in the hippocampus.Our results show that densinReceived Sept.2,2009;revised Feb.12,2010;accepted Feb.19,2010.This work was supported by grants from the National Institutes of Health(HL087120,DC009433to A.L.;MH63232toR.J.C.;NS054614toY.M.U.;andT32HL07121toC.J.C.),theAmericanHeartAssociation(A.L.),theIsraelBinational Science Foundation(A.L.),and the Austrian Science Fund(P17807-B05to G.J.O.).We thank Diane Lips-combe and Randy Hall for cDNAs,Jo¨rg Striessnig for Ca v1.3knock-out mice,and Irina Calin-Jageman for help ingenerating the HA-tagged Ca v1.3.*M.A.J.and C.J.C.contributed equally to this work.Correspondence should be addressed to Amy Lee,Department of Molecular Physiology and Biophysics,Univer-sity of Iowa,5-611Bowen Science Building,51Newton Road,Iowa City,IA52242.E-mail:amy-lee@.DOI:10.1523/JNEUROSCI.4367-09.2010Copyright©2010the authors0270-6474/10/305125-11$15.00/0The Journal of Neuroscience,April14,2010•30(15):5125–5135•5125functionally recruits CaMKII to Ca v1.3channels,which causes frequency-dependent facilitation of Ca v1.3Ca2ϩsignals that may regulate neuronal excitability.Materials and MethodscDNAs.Ca v1.3channels consisted of rat␣11.3(containing exon42, GenBank#AF3700010,in pCDNA6from D.Lipscombe,Brown Univer-sity,Providence,RI),␤1b(GenBank#NM017346),and␣2␦(GenBank #M21948).The following cDNAs were described previously:FLAG-andGST-tagged␣11.3constructs(Calin-Jageman et al.,2007);CaMKII␣, CaMKII␣T286A(Brickey et al.,1990;McNeill and Colbran,1995);GFP-densin and GFP-densin⌬483-1377(Jiao et al.,2008);His-densin PDZ (Fam et al.,2005);and p␤A-eGFP(Obermair et al.,2004).For GFP-densin⌬PDZ,the PDZ domain-encoding region(aa1452-1542)was deleted by PCR amplification and ligation into Bgl II and Sac II sites of pEGFP(Clontech,BD BioSciences).For external hemagglutinin(HA)epitope-tagged␣11.3,the sequence for the HA tag was cloned into the extracellular loop connecting IIS5-IIS6 according to a similar strategy for␣11.2described previously(Altier et al., 2002).The insertion of the HA tag had no effects on channel properties as assessed by electrophysiological recordings of transfected HEK293T cells (data not shown).To facilitate neuronal expression HA-␣11.3was sub-cloned into the p␤A-PL expression vector(Obermair et al.,2010)in a two-step procedure.First,a Hin dIII–Sal I fragment(3303bp)containing the5Јcoding sequence of␣11.3was cloned into the corresponding re-striction sites of p␤A-PL.Second,the BsiW I–Sac II fragment(6019bp)of HA-␣11.3was ligated together with a25bp Sac II–Spe I linker into the BsiW I and Xba I sites of the intermediate construct,eliminating Spe I and Xba I recognition sequences and yielding p␤A-HA-␣11.3.The construct was verified by sequencing before use(MWG-Biotech). Antibodies.Goat␣11.3antibodies(Calin-Jageman et al.,2007)and goat densin antibodies(Ab650)(Jiao et al.,2008)were characterized previously.Rabbit␣11.3antibodies(Ab144)were raised against a synthetic peptide corresponding to␣11.3N-terminal sequence (MQHQRQQQEDHANEANYARGTRKC;Covance Research Prod-ucts).Characterization of Ab144specificity is described in supplemental Figure1,available at as supplemental material. Briefly,by immunofluorescence and Western blot,these antibodies la-beled HEK293T cells transfected with Ca v1.3but not untransfected cells (Fig.S1A,B,available at as supplemental material). In addition,Ab144recognized a protein consistent in size with␣11.3in hippocampal lysates of wild-type but not Ca v1.3knock-out mice(pro-vided by Jo¨rg Striessnig,University of Innsbruck,Innsbruck,Austria) (Fig.S1C,available at as supplemental material). Other antibodies used were as follows:mouse monoclonal antibodies against CaMKII␣(Affinity Bioreagents),FLAG(Sigma-Aldrich),and GFP(Santa Cruz Biotechnology);and rabbit polyclonal antibodies against densin(Santa Cruz Biotechnology)and hexahistidine(anti-His) antibodies(Santa Cruz Biotechnology).For immunofluorescence of cultured neurons,the following antibod-ies were used:rat anti-HA(monoclonal,clone3F10,Roche Diagnostics,1:100),mouse anti-PSD-95(monoclonal,clone6G6–1C9,AffinityBioreagents,1:1000),rabbit polyclonal anti-GFP(1:20,000;Invitrogen),goat anti-rabbit Alexa488(1:2000),goat anti-mouse Alexa594(1:4000),and goat anti-rat Alexa594(Invitrogen,1:4000).Cell culture and transfection.Human embryonic kidney cells(HEK293)or HEK293cells transformed with SV40T-antigen(HEK293T)were maintained in DMEM with10%fetal bovine serum(Invitrogen)at37°C in a humidified atmosphere with5%CO2.Cellswere grown toϳ80%confluence and transfected using Gene Porterreagent(Gene Therapy Systems)or Fugene(Roche).For pull-down as-says,cells were transfected with GFP-densin(6␮g).For coimmunopre-cipitation of GFP-densin and Ca v1.3,cells were transfected with cDNAs encoding Ca v1.3[FLAG-␣11.3(6␮g),␤1b(2␮g),and␣2␦(2␮g)]with or without GFP-densin(4␮g).For coimmunoprecipitation of CaMKII and GFP-densin or⌬483-1377,cells were transfected with GFP-densin(7␮g) or GFP-⌬483-1377(2␮g)and CaMKII␣(1␮g).For electrophysiology,HEK293T cells were transfected with␣11.3(1.5␮g),␤1b(0.5␮g),and ␣2␦(0.5␮g)with or without GFP-tagged densin(0.5␮g)and/or CaMKII (0.5␮g).Electrophysiological recordings.At least48h after transfection,whole-cell patch-clamp recordings of transfected cells were acquired with a HEKA Elektronik(Lambrecht/Pfalz)EPC-8or EPC-9patch-clamp am-plifier.Data acquisition and leak subtraction using a P/4protocol were performed with Pulse software(HEKA Elektronik).Extracellular record-ing solutions contained the following(in m M):150Tris,1MgCl2,and10 CaCl2or10BaCl2.Intracellular solutions contained the following(in m M):140N-methyl-D-glucamine,10HEPES,2MgCl2,2Mg-ATP,and5 EGTA or10BAPTA.The pH of the recording solutions was7.3,adjusted with methanesulfonic acid.Electrode resistances were3–4M⍀in the bath solution.Series resistance was compensated up to80%.Igor Pro software(Wavemetrics)was used for data analysis.Except for Fig.7B below,data analysis was restricted to Ca v1.3currents with amplitudes of Ͼ250pA.All averaged data are presented as the meanϮSEM.Statistical significance was determined with either Student’s t test or ANOVA with post hoc analyses,as indicated(SigmaPlot;Systat Software).Binding assays.GST-and His-tagged fusion proteins were prepared as described previously(Robison et al.,2005;Calin-Jageman et al.,2007). For pull-down assays,GST-␣11.3CT was immobilized on glutathione agarose beads and incubated with lysate from GFP-densin-transfected HEK293T cells in binding buffer[Tris-buffered saline(TBS;50m M Tris-HCl,pH7.5,150m M NaCl),0.1%Triton X-100,and protease inhibitors (1mg/ml each of PMSF,pepstatin,aprotinin,and leupeptin)].Binding reactions proceeded at4°C for90min.Beads were washed three times with binding buffer(1ml)at4°C,and bound proteins were eluted,re-solved by SDS-PAGE,and transferred to nitrocellulose.Western blotting was performed with appropriate antibodies followed by HRP-conju-gated secondary antibodies and enhanced chemiluminescent detection reagents(GE Healthcare).For overlay assays,GST-␣11.3CT,GST-␣11.3 C-terminal leucine to alanine(CT L-A),or GST(1␮g)was run on a 4–20%SDS-polyacrylamide gel and transferred to nitrocellulose.The membrane was blocked in blocking buffer(2%milk,TBS,0.1%Tween 20)for30min(4°C)before incubation with His-tagged densin-PDZ domain(300n M in blocking buffer)for1h at4°C.Bound protein was detected by Western blotting with anti-His antibodies. Coimmunoprecipitation assays.For coimmunoprecipitation from mouse hippocampus,a Triton X-100-soluble fraction(0.5ml)was pre-pared as described previously(Abiria and Colbran,2010)and incubated with10␮g of either goat IgG or affinity-purified goat antibodies that recognize CaMKII,densin,or␣11.3.After1h,10␮l of protein-G Sepha-rose(GE Healthcare Bio-Sciences)was added and the incubation contin-ued forϳ2h at4°C.The resin was rinsed three times in1ml of solubilization buffer and bound proteins were analyzed by SDS-PAGE and Western blotting with mouse antibodies to CaMKII␣or rabbit an-tibodies to densin and␣11.3(Ab144).For coimmunoprecipitation of GFP-densin and␣11.3,transfected HEK293T cells were solubilized in lysis buffer(50m M Tris-HCl,pH7.5, 150m M NaCl,1%NP-40,0.25%sodium deoxycholate,1m M EDTA,and protease inhibitors),incubated at4°C for30min,and subjected to cen-trifugation at100,000ϫg(30min)to remove insoluble material.The supernatant was incubated with5␮g␣11.3antibodies and50␮l of pro-tein A-Sepharose(50%slurry)for4h,rotating at4°C.After three washes with RIPA buffer(1ml),proteins were eluted with SDS-containing sam-ple buffer and subjected to SDS-PAGE.Coimmunoprecipitated proteins were detected by Western blotting with specific antibodies as indicated. For coimmunoprecipitation of CaMKII and densin or⌬483-1377, transfected HEK293cells were lysed on ice with lysis buffer[2m M Tris-HCl,pH7.5,1%(v/v)Triton X-100,0.1m M PMSF,1m M benzamidine, 5mg/L leupeptin,20mg/L soybean trypsin inhibitor].After sonication (2ϫ5s),lysates were incubated at4°C for30min and then centrifuged for15min at10,000ϫg.NaCl was added into the supernatant with the final concentration as150m M and equal aliquots of the supernatants were incubated with10␮g of densin Ab450or goat IgG,or goat CaMKII␣antibody overnight at4°C.After addition of GammaBind5126•J.Neurosci.,April14,2010•30(15):5125–5135Jenkins et al.•Cav1.3Regulation by Densin and CaMKIIPlus-Sepharose (30␮l of 50%slurry;GE Healthcare),incubations were continued for 2h at 4°C.Beads were collected by centrifugation and washed at least five times with 1ml of wash buffer containing 50m M Tris-HCl,pH 7.5,1%(v/v)Triton X-100,and 150m M NaCl.Immune complexes were solubilized in SDS-PAGE sample buffer before elec-trophoresis and Western blotting.Interpretations of results from co-immunoprecipitation and binding assays were based on at least three independent experiments.Ca v 1.3and densin targeting in transfected mouse hippocampal neurons.Low-density cultures of hippocampal neurons were prepared from 16.5-d-old embryonic BALB/c mice as described previously (Obermair et al.,2004).The following plasmids (1.5␮g total DNA)were transfected into neurons on day 6using Lipofectamine 2000transfection reagent (In-vitrogen):GFP-densin (single transfection)and p ␤A-eGFP and p ␤A-HA-␣11.3(cotransfection).Cells were immunostained and analyzed 11–14d after transfection.For double-immunolabeling (GFP-densin and PSD-95)neurons were fixed with methanol for 10min at Ϫ20°C and rehydrated in PBS at room temperature.Fixed neurons were incubated in 5%normal goat serum in PBS,0.2%bovine serum albumin,and 0.2%Triton X-100(PBS/BSA/Triton)for 30min.Primary antibodies were applied in PBS/BSA/Triton at 4°C overnight and detected by fluorochrome-conjugated secondary antibodies.For staining of surface-expressed HA-␣11.3,living neurons were incubated with the rat anti-HA antibody for 30min at 37°C.Then the cultures were rinsed in Hank’s buffered saline,fixed in 4%paraformaldeyde/4%sucrose for 10min,blocked with 5%normal goatserum in PBS/BSA/Triton,and incubated with the secondary antibody for 1h (Obermair et al.,2010).Coverslips were then washed and mounted in p-phenylene-diamine-glycerol to retard photobleaching.Preparations were analyzed on an AxioImager microscope (Carl Zeiss)using a 63ϫ, 1.4numerical aperture (NA)objective.Images were re-corded with a cooled CCD camera (SPOT;Diagnostic Instruments)and Metavue image processing software (Universal Imaging).Composite images were arranged in Adobe Photoshop 9(Adobe Systems)and linear ad-justments were performed to correct black level and contrast.Measurements of intracellular Ca 2ϩconcen-tration ([Ca 2ϩ]i ).HEK293T cells transfected with Ca v 1.3,densin,and CaMKII were loaded with fura-2(Invitrogen)via the patch pipette (100␮M ),which also contained the intracellu-lar recording solution described for electro-physiological recordings.Cells were placed in a flow-through chamber mounted on the stage of an inverted IX-71microscope (Olympus).Fluorescence was alternately excited at 340nm (12nm bandpass)and 380nm (12nm band-pass)using the Polychrome IV monochroma-tor (TILL Photonics)via a 40ϫoil-immersion objective (NA ϭ1.35,Olympus).Emitted fluorescence was collected at 510nm (80nm bandpass)using an IMAGO CCD camera (TILL Photonics).Pairs of 340/380nm im-ages were sampled at 10Hz.Fluorescence was corrected for background,as determined in an area that did not contain a cell.Data were processed using TILLvisION 4.0.1.2(TILL Photonics)and presented as a fluores-cence ratio of F 340/F 380,where F 340and F 380are fluorescence intensities at the excitation wavelengths 340and 380nm,respectively.Averaged data are presented as the mean ϮSEM and were statistically compared by t test.ResultsCaMKII does not affect Ca v 1.3properties in transfected HEK293T cellsBecause of the importance of CaMKII as both regulator and transducer of Ca v 1Ca 2ϩsignals (Dzhura et al.,2000;Wheeler et al.,2008),we tested whether CaMKII directly influences Ca v 1.3function.For this purpose,we compared channel properties in HEK293T cells transfected with Ca v 1.3alone (␣11.3,␤1b ,and ␣2␦)and those cotransfected with Ca v 1.3and CaMKII ␣.This isoform of CaMKII was chosen since it is one of the major iso-forms of CaMKII in the brain (Colbran and Brown,2004)and cannot be detected endogenously in HEK293T cells (Fig.1A ).We found that CaMKII had no effect on voltage-dependent activa-tion of Ca v 1.3Ba 2ϩcurrents (I Ba ).Parameters describing cur-rent–voltage (I–V )curves were not different in cells with Ca v 1.3and those with Ca v 1.3plus CaMKII (p ϭ0.32for k ,p ϭ0.92for V 1/2,by t test)(Fig.1B ).Expression of CaMKII also did not affect mean Ca v 1.3current amplitudes (535Ϯ78pA for Ca v 1.3alone,n ϭ18vs 844Ϯ227pA for plus CaMKII,n ϭ16;p ϭ0.18by t test).While CaMKII enhances VDF of Ca v 1.2(Lee et al.,2006),we did not find the same result for Ca v 1.3.VDF was measured as the ratio of the amplitude of I Ba evoked before or after a conditioning prepulse.With this protocol,I BaFigure 1.CaMKII does not affect Ca v 1.3activation or voltage-dependent facilitation.A ,Western blot with CaMKII antibodies showingCaMKIIexpressioninHEK293TcellstransfectedwithCaMKII ␣aloneor ϩCa v 1.3(␣11.3,␤1b ,␣2␦)butnotuntransfected cells(U).B ,I Ba wereevokedby50mstestpulsesfrom Ϫ90tovariousvoltagesinHEK293TcellstransfectedwithCa v 1.3alone(n ϭ18,open circles)or cotransfected with CaMKII (n ϭ16,closed circles).Representative current traces (left)and normalized (Norm.)I–V relationship (right)are shown.For Ca v 1.3alone,V 1/2ϭϪ26.3Ϯ2.4mV,k ϭϪ6.7Ϯ0.5.For Ca v 1.3plus CaMKII,V 1/2ϭϪ26.6Ϯ1.5mV,k ϭϪ7.2Ϯ0.3.C ,I Ba was evoked by 10ms test pulse from Ϫ90to Ϫ20mV before (P1)and after (P2)a 20ms prepulse (Pre)to various voltages.Left,Voltage protocol and representative test currents using ϩ60mV prepulse.Dashed line indicates initial P1current amplitude.Right,Facilitation was measured as the ratio of the P2and P1test currents and plotted against prepulse voltage for cells transfected with Ca v 1.3alone (n ϭ10)or cotransfected with CaMKII (n ϭ12).By two-way ANOVA,there was no difference in facilitation between groups.Jenkins et al.•Ca v 1.3Regulation by Densin and CaMKIIJ.Neurosci.,April 14,2010•30(15):5125–5135•5127underwent modest VDF that was not further affected by CaMKII (p ϭ0.68)(Fig.1C ).We also did not observe any differences in the extent of I Ba inactiva-tion either during sustained or repeti-tive stimuli (data not shown).Densin binds to Ca v 1.3␣1C terminus In contrast to our findings,previous stud-ies of SH-SY5Y human neuroblastoma cells and cortical neurons implicated CaMKII and release of Ca 2ϩfrom intra-cellular stores in the potentiation of Ca V 1.3currents at negative voltages following stim-ulation with insulin-like growth factor 1(Gao et al.,2006b).Analogous to the role of cAMP-dependent protein kinase (PKA)anchoring proteins for PKA regulation of Ca v 1channels (Hulme et al.,2004),feed-back modulation of Ca v 1.3by CaMKII may require additional adaptor proteins present in neurons but not HEK293T cells.Densin was considered since it binds to CaMKII (Strack et al.,2000;Walikonis et al.,2001)and contains a type I PDZ domain that could associate with the cor-responding recognition site at the distalCT of ␣11.3(Fig.2A ).Consistent with this possibility,GFP-tagged densin coimmu-noprecipitated with FLAG-tagged ␣11.3in HEK293T cells (Fig.2B )and bound in vitro to GST-tagged proteins containing the ␣11.3CT,but not GST (Fig.2C ).To test the importance of the PDZ-binding sequence of ␣11.3for the interaction,we mutated the CT L-A ,which should prevent PDZ binding (Songyang et al.,1997;Calin-Jageman et al.,2007).Unlike for the wild-type ␣11.3CT,the densin PDZ do-main did not bind to CT L-A (Fig.2D ).These results confirmed a direct interaction of densin with the␣11.3CT PDZ-binding sequence.We next investigated the potential for densin and Ca v 1.3tointeract in neurons.We first tested whether Ca v 1.3channels anddensin were associated with the same subcellular compartmentsin neurons.To restrict analysis to plasma membrane channels,weanalyzed the localization of transfected HA-tagged ␣11.3in whichthe HA tag was inserted in an extracellular domain of ␣11.3.Immunofluorescence with HA antibodies applied to live neuronscotransfected with eGFP and HA-␣11.3revealed a punctate dis-tribution for Ca v 1.3along the shaft and spines throughout thedendritic arbor (Fig.3A ).GFP-tagged densin showed a similardistribution,which was predominantly postsynaptic as indicatedby colocalization with PSD-95(Fig.3B ).Unfortunately,we could not determine whether both GFP-densin and Ca v 1.3were colocalized since cotransfection of thecorresponding cDNAs was deleterious to neuronal survival (datanot shown).Therefore,we tested for a physical interaction be-tween densin and Ca v 1.3by coimmunoprecipitation.The ␣11.3antibodies,but not control IgG,coimmunoprecipitated densinwith ␣11.3from solubilized mouse hippocampal membrane ex-tracts (Fig.3C ).In the reverse approach,␣11.3was similarlybrought down by densin antibodies (Fig.3D ).Consistent with arole for densin in scaffolding CaMKII to the channel complex,CaMKII was coimmunoprecipitated regardless of whether anti-bodies against densin or ␣11.3were used (Fig.3C ,D ).Moreover,CaMKII antibodies specifically coimmunoprecipitated both den-sin and ␣11.3(Fig.3E ),despite a weak nonspecific interaction of CaMKII with the control IgG,which was most likely due to abun-dance of CaMKII in hippocampal extracts.Collectively,these results support the existence of a ternary complex comprised of densin,CaMKII,and Ca v 1.3channels in the hippocampus.Densin and CaMKII cause Ca 2؉-dependent facilitation of Ca v 1.3Ca 2؉currents To test whether densin may functionally recruit CaMKII for modulation of Ca v 1.3,we analyzed the effect of cotransfecting densin plus CaMKII with Ca v 1.3in HEK293T cells.While densin plus CaMKII did not affect voltage-dependent activation or facil-itation of Ca v 1.3(data not shown),they significantly increased the amplitude of Ca v 1.3Ca 2ϩcurrents (I Ca )during trains of depolarizations (100Hz)(Fig.4A ).With this voltage protocol,I Ca in cells transfected with Ca v 1.3alone inactivates rapidly (ϳ40%within 50ms)(Fig.4A ),due to Ca 2ϩ-dependent inacti-vation (CDI)mediated by calmodulin (Yang et al.,2006).In cells cotransfected with densin plus CaMKII,I Ca inactivated signifi-Figure2.Densinbindsto ␣11.3.A ,Top,Rat ␣11.3withC-terminaltypeIPDZ-domainindicatedwithitalics;bottom,ratdensin with leucine-rich region (LRR),CaMKII-binding domain (CBD),and PDZ domain (not drawn to scale).Numbers indicate amino acidboundaries.B ,Coimmunoprecipitation of GFP-densin with FLAG-tagged ␣11.3.Lysates from HEK293T cells cotransfected withCa v 1.3(FLAG-␣11.3,␤1b ,␣2␦)and GFP-densin (lane 1)or transfected with GFP-densin alone (lane 2)were incubated with goat␣11.3antibodies.Immunoprecipitated proteins were detected by Western blotting with FLAG (top)or GFP (bottom)antibodies.Asterisk indicates nonspecific band detected by GFP antibody.C ,Pull-down of GFP-tagged densin by GST-tagged ␣11.3CT (GST-CT).GST (lane 2)or GST-CT (lane 3)was immobilized on glutathione-agarose beads and incubated with lysates from HEK293T cells transfected with GFP-densin.Top,Western blot with GFP antibody.Bottom,GST-CT and GST protein used for pull-down are indicated in the Ponceau-stained ne 1(In)shows ϳ5%of the GFP-transfected cell lysate used in the assay.D ,Binding of His-tagged densin PDZ-domain to GST-CT but not to GST or CT with mutation in PDZ-binding sequence (GST-CT L-A )in protein overlay assay.GST proteins were separated by SDS-PAGE and transferred to nitrocellulose,which was incubated with His-densin.Left,Ponceau staining shows levels of GST proteins used in the assay.Right,Western blot with anti-His antibodies shows binding of His-densin PDZ only to GST-CT.5128•J.Neurosci.,April 14,2010•30(15):5125–5135Jenkins et al.•Ca v 1.3Regulation by Densin and CaMKIIcantly less,with amplitudes at the end of the 300ms train that were ϳ45%greater than in cells with Ca v 1.3alone (Fig.4A ,C ).The enhancement of I Ca was independent of changes in voltage-dependent activation or peak current amplitude,which were not different in cells transfected with Ca v 1.3alone (k ϭϪ12.0Ϯ1.0;V 1/2ϭϪ7.1Ϯ0.3;I Ca amplitude at Ϫ10mV ϭ1298.9Ϯ304.6pA;n ϭ9)and Ca v 1.3plus densin plus CaMKII (k ϭϪ12.5Ϯ2.2,p ϭ0.68;V 1/2ϭϪ7.2Ϯ0.5,p ϭ0.34;I Ca amplitude at Ϫ10mV ϭ649.4Ϯ165.2pA,p ϭ0.21;n ϭ10;by t test).To determine whether the effect of densin plus CaMKII was Ca 2ϩdepen-dent,we analyzed I Ba .Because Ba 2ϩdoes not support Ca 2ϩ-dependent inactiva-tion,the amplitude of I Ba remains rela-tively constant throughout the train (Fig.4B ).In contrast to effects on I Ca ,densin plus CaMKII modestly increased inactiva-tion of I Ba (ϳ11.8%)(Fig.4B ,C ).The ef-fects of densin plus CaMKII were not seen when densin or CaMKII was singly co-transfected with Ca v 1.3(Fig.4C ).These results reveal that Ca v 1.3currents undergo CDF during repetitive stimuli,which re-quires both densin and CaMKII.Densin binding to the ␣11.3CT and CaMKII is required for CDF of Ca v 1.3Since densin binds both ␣11.3CT (Fig.2C ,D )and CaMKII (Strack et al.,2000;Walikonis et al.,2001),we hypothesized that CDF requires densin to scaffold CaMKII to the ␣11.3CT.If so,then pre-venting these interactions should block CDF.We tested this prediction first with ␣11.3containing the L-A mutation,which prevents densin binding (Fig.2D ).As ex-pected,there was no significant effect of densin plus CaMKII on I Ca inactivation for channels containing the L-A mutation (p ϭ0.15)(Fig.5A ).We next examined the effect of deleting the PDZ domain from densin (⌬PDZ),which should also prevent binding to ␣11.3CT.Unlike full-length densin,the ⌬PDZ truncation did not affect I Ca amplitude at the end of thetrain (p ϭ0.82compared with Ca v 1.3alone)(Fig.5B ).Together,these data con-firm the requirement for densin binding to ␣11.3CT for CDF.CaMKII directly interacts with the C-terminal domain of densin in vitro (Fig.2A )(Strack et al.,2000;Walikonis et al.,2001)and this interaction is sufficient for coimmunoprecipitation of CaMKII withdensin from transfected HEK293cells (Jiao et al.,2008).In ongoing studies to define domains in full-length densin thatare necessary for interaction with CaMKII,we found that a large internal deletion (⌬483-1377)in a naturally occurring densinsplice variant substantially reduces the coimmunoprecipitation of CaMKII when compared with the full-length densin (Fig.5C ).Apparently,the deleted region (⌬483-1377)is required forCaMKII binding.Since the ⌬483-1377densin variant retainstheFigure 3.Densin and Ca v 1.3are targeted to dendritic spines in neurons and form a ternary complex with CaMKII in the hippocampus.A ,B ,Epifluorescence images of immunofluorescently labeled transfected mouse hippocampal neurons in low-density culture.Grayscale images show fluorescence due to individual fluorophores;merged images are shown in color with regions of colocalization appearing yellow.A ,Dendritic tree of neuron [17days in vitro (DIV)]cotransfected with HA-␣11.3and eGFP after live-cell staining with HA antibody (anti-HA).HA-␣11.3-positive puncta (left,red)are present on the neuronal surface throughout the dendrites.A dendrite segment shown at higher magnification (right)reveals HA-␣11.3puncta on the plasma membrane of dendritic spines (arrowheads).B ,Neuron (20DIV)transfected with GFP-densin,double labeled with antibodies against GFP (anti-GFP,green)and the postsynaptic density protein 95(anti-PSD95,red).Immunofluorescence for GFP was re-quiredduetotheweakintrinsicfluorescenceofGFP-densin.GFP-densinandPSD95displaysimilarclustereddistributionsthrough-out the dendrites.At higher magnification (right),there is strong colocalization of GFP-densin (green)with PSD95(red;arrowheads).Scalebars:left,20␮m;right,5␮m.C–E ,Coimmunoprecipitationofdensin,CaMKII,and ␣11.3.Mousehippocampallysates were incubated with control goat IgG or affinity purified goat antibodies against ␣11.3(C ),densin (D ),or CaMKII (E ).Coimmunoprecipitated proteins are indicated by arrows in Western blots for ␣11.3(top),densin (middle),or CaMKII (bottom).Input lanes (In)represent 5%of lysates used in the assay.Jenkins et al.•Ca v 1.3Regulation by Densin and CaMKIIJ.Neurosci.,April 14,2010•30(15):5125–5135•5129。